The present invention relates to a method of assay for antigen, specifically to a method of assay for antigen which permits assay for low molecular antigens (including haptens) with high sensitivity.
Assay of antigen in biocomponents, particularly those in human body fluids, is very important in clinical situations. It is widely used for endocrinologic examinations by hormone measurement, cancer diagnosis, quantitative determination of drugs and other purposes.
Microdetermination for antigens as mentioned above has conventionally been achieved by immunoassay methods, such as RIA, EIA and FIA.
The immunoassay methods can be roughly classified into two groups, one based on the competitive method and the other based on the sandwich method.
Immunoassay was first established as RIA based on the competitive method by Yallow et al. The competitive method is an assay method utilizing the inhibition of the binding of labeled antigen to antibody by the antigen to be assayed. When the amounts of the labeled antigen and antibody added are increased, the inhibition by a small amount of antigen to be assayed becomes undetectable. When the amounts of added labeled antigen and antibody are reduced, the ratio of labeled antigen bound to the antibody decreases, thus posing a limitation. Therefore, the detection limit is normally about 1 fmol (10.sup.-15 mol)
On the other hand, the sandwich method is an assay method in which the antigen to be assayed is bound with both an antibody-coated carrier and a labeled antibody in a sandwich state. In this method, the detection limit is 100 to 1000 times superior to that of the competitive method, thus permitting assay at the amol (10.sup.-18 mol) level. It has recently become common to assay protein hormones, such as TSH and hCG, by sandwich methods known as ELISA and IRMA.
However, there are some limitations posed on the sandwich methods as to measurable antigens. Specifically, the antigen to be assayed by the sandwich method needs to have at least two epitopic sites at a time at which the antigen can bind to the antibody, and low molecular antigens cannot be assayed by the sandwich method.
Also, even when the antigen is theoretically measurable by the sandwich method, it is difficult to obtain two antibodies against the two epitopic sites in some cases; in others, even when two antibodies against the two epitopic sites can be obtained, high titer antibody might be obtained at only one epitopic site, which hampers high sensitivity assay.
The object of the present invention is to provide a method of assay for antigen with high sensitivity based on characteristic features of the sandwich method, using only one antibody against a single epitopic site, specifically a method which permits assay for low molecular antigens (haptens), such as peptides, steroids and drugs, which have never been assayed by the conventional sandwich method, with sensitivity as high as that of the sandwich method.